Autoantibodies are a characteristic of most autoimmune diseases and appear in the serum many years before the onset of clinical disease, suggesting an early break in B cell tolerance. An important role for B cells in some autoimmune diseases has been supported by successful treatment of patients with anti-CD20 monoclonal antibodies that eliminate B cells. The underlying mechanisms that account for autoreactive B cell and autoantibody production in autoimmune diseases are poorly defined. We analyzed B cell tolerance in untreated active rheumatoid arthritis (RA) and type 1 diabetes (T1D) patients by testing the specificity of recombinant antibodies cloned from single new emigrant and mature naive B cells. RA and T1D patients exhibit defective central and peripheral B cell tolerance checkpoints that result in the accumulation of selfreactive mature naive B cells, likely contributing to pathogenesis. The long range goal of the proposed research is to determine the mechanisms that regulate B cell tolerance in healthy humans but may be defective in patients with autoimmune diseases. The working hypothesis is that developing autoreactive B cells in patients with autoimmune diseases surfer from intrinsic defects that impinge on their proper counterselection in the bone marrow. In addition, defects in regulatory T cell functions and elevated BAFF levels may alter peripheral B cell tolerance and result in the abnormal recruitment and activation of autoreactive B cell clones. The first aim of the project will consist of determining whether B cell tolerance defects in T1D persist after B cell depletion and may therefore predict patients' relapse. The second part of the project will analyze the development and involvement of autoreactive B cells in primary Sjogren's syndrome. The third part of the project will characterize the molecular basis for early defects in B cell tolerance checkpoints by analyzing the impact of known polymorphism that are protective or associated to the development of autoimmune diseases.